Induced chromosome doubling in plants

ABSTRACT

Methods to generate doubled haploid plants and plant components using low mammalian toxicity chromosome doubling agents are disclosed. Chromosome doubling agents provide low mortality rates and higher chromosome doubling rate in plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14,037,501 filed Sep. 26, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/646,064, filed Dec. 23, 2009, pending, thedisclosures of which are hereby incorporated herein by this reference intheir entirety.

FIELD

The present invention generally relates to plant breeding and generatingdoubled haploid plants.

BACKGROUND

Traditional plant breeding to include parental lines for desirabletraits involves crossing selected parental lines to introduce thosedesirable traits into the progeny of the cross. In a crossing-basedapproach, often, not only the desirable trait is transferred to theprogeny but some randomization of the genomes of both parental linesoccurs. This results in a wide segregation and variation of morphologyand other traits of the progeny, which are not predictable. Theuncontrolled variation renders the progeny selection process very long,cumbersome and laborious especially if the desired traits are notexpressed early in the progeny or if the desired trait is recessive.

In an effort to minimize the random variation, breeders preferhomozygous parental lines (inbreds) so that the genetic make up of theF₁ generation is more predictable. The inbreds with a desirable traitare generated by back-crossing a heterozygote with its parental lines,followed by segregation selection and repeated back-crossing. However,this repeated back-crossing is also very long, usually up to 6 to 7times, depending on the plant, would produce a homozygous plant with thedesired trait. Of course, the time scale involved here is dictated bythe rate at which plants grow to maturity and set seed and several yearscan be necessary to produce the desired homozygous parent line.

Haploid plants contain one half of the usual complement of genes. Normalplants are diploid in that they have two complete sets of chromosomes,one from each parent. Polyploid plants have more than two sets ofchromosomes. Haploid plants are capable of growing to maturity but aregenerally sterile. There are several known methods of generating haploidplants.

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple back-crossings is to produce haploids and then doublethe chromosomes to form doubled haploids. The production of doubledhaploid plants yields highly uniform inbred lines and is especiallydesirable as an alternative to sexual inbreeding of longer-generationcrops. By producing doubled haploid progeny, the number of possible genecombinations for inherited traits is more manageable. Thus, an efficientdoubled haploid technology can significantly reduce the time and thecost of inbred and cultivar development. Chemicals, such as colchicinesare used as chromosome doubling agents. However, due to their mammaliantoxicity and low efficiency, their use in plant breeding has beenlimited. Therefore, agents to induce chromosome doubling in plants withlow mammalian toxicity are desirable.

SUMMARY

Methods for generating doubled haploid plants, seeds, cells, and cellcultures are provided. The methods disclosed herein, increase theefficiency of the doubled haploid generation by increasing the number ofdoubled haploids obtained, exhibit low mammalian toxicity and reducedthe time required to produce the doubled haploids.

DETAILED DESCRIPTION OF THE INVENTION

Methods to obtain doubled haploid plants or plant components using lowmammalian toxicity chromosome doubling agents are disclosed. Contactinga haploid plant component, e.g., whole plant or a collection orpopulation of plant cells with low mammalian toxicity chromosomedoubling agents results in the generation of doubled haploid plants orplant components.

Chemicals listed in the U.S. Pat. No. 5,866,513 to Michelotti et al.,are used to generate doubled haploid plants. The disclosure of U.S. Pat.No. 5,866,513 is incorporated herein by reference in its entirety. Forexample, Table I and Ia on Cols. 3 and 4, 5 and 6, and 7 and 8 of U.S.Pat. No. 5,866,513, list about 76 specific example compounds and each ofthose compounds is herein incorporated by reference in its entirety. Inaddition, methods of making some of the specific examples as describedin Cols. 11-22 are incorporated herein by reference in its entirety.

In one embodiment a method is described for generating doubled haploidplant cells comprising contacting a population of haploid plant cellswith a composition comprising a low mammalian toxicity chromosomedoubling agent of following formula:

wherein R¹, R², R³, R⁴, and R⁵ are in each instance independentlyselected from the group consisting of hydrogen, alkoxy, nitro, halo,alkyl, and haloalkyl; A and B are each independently CH₂, CHR^(a) orCR^(a)R^(b), wherein R^(a) and R^(b) are in each instance independentlyselected from the group consisting of hydrogen, halogen, cyano, alkyl,alkenyl, alkoxy, haloalkyl, haloalkoxyl, alkylcarbonyl andalkoxycarbonyl, or R^(a) and R^(b) together with the attached carbonform a cycloalkylene; Z is CH═CH, CH₂—CH₂, CH₂═CH(CH₃), orCR^(d)═CR^(e), where R^(d) and R^(e) are each independently hydrogen,alkyl or R^(d) and R^(e) together with the attached carbons form acycloalkylene; X and Y are each independently selected from the groupconsisting of hydrogen, NO₂, CN, halogen, alkylcarbonyl, alkoxycarbonyl,and CH₂—NO₂, provided that X and Y are not both hydrogen; anddiastereomers and stereoisomers thereof.

In a preferred embodiment a method is described for generating doubledhaploid plant cells comprising contacting a population of haploid plantcells with a composition comprising a low mammalian toxicity chromosomedoubling agent of following formula:

wherein R¹, R², R³, R⁴, and R⁵ are in each instance independentlyselected from the group consisting of hydrogen, alkoxy, nitro, halo,alkyl, and haloalkyl; Z is CH═CH, CH₂—CH₂, CH₂═CH(CH₃), orCR^(d)═CR^(e), where R^(d) and R^(e) are each independently hydrogen,alkyl or R^(d) and R^(e) together with the attached carbons form acycloalkylene; X and Y are each independently selected from the groupconsisting of hydrogen, NO₂, CN, halogen, alkylcarbonyl, alkoxycarbonyl,and CH₂—NO₂, provided that X and Y are not both hydrogen; anddiastereomers and stereoisomers thereof.

In one embodiment the low mammalian toxicity chromosome doubling agentcomprises a compound of the following formula and diastereomers andstereoisomers thereof:

Suitable dosage for the low mammalian toxicity chromosome doublingagents for the seedling soak method, disclosed herein, include, forexample, 0.01 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90μM, 100 μM, 125 μM, 150 μM, 200 μM, 500 μM, and 1000 μM. Suitable rangesalso include, for example, 0.1-10 μM, 1-100 μM, 5-125 μM, 25-200 μM,50-500 μM, 15-150 μM and 1-10,000 μM.

The low seedling mortality of the chromosome doubling agents, disclosedherein, when compared to colchicine (e.g., at 0.06% w/v) can range, forexample, from less than 10% to about 40% or less than about 5% to about20% or less than about 15% to about 25% or less than 50% of the totalnumber of seedlings or plant cells treated.

Suitable dosage for the low mammalian toxicity chromosome doublingagents for the seedling foliar application method, disclosed herein,include, for example, 3.5 g ai/ha, 70 g ai/ha, 140 g ai/ha, 280 g ai/ha.Suitable application rates ranges include, for example, 5 g ai/ha to1120 g ai/ha, and more preferably to 2,800 g ai/ha.

The low mammalian toxicity of the chromosome doubling agents, disclosedherein, may show LD₅₀ (rat) that is greater than or equal to 400 mg/kgor 500 mg/kg or 600 mg/kg or 700 mg/kg or 800 mg/kg or 900 mg/kg or 1000mg/kg. Suitable LD₅₀ (rat) range may also include 200-1000 mg/kg or100-1000 mg/kg or greater than 100 mg/kg or greater than 1000 mg/kg.

A haploid plant has a single set of chromosomes and the reduced numberof chromosomes (n) in the haploid plant is equal to that in the gamete.For example, a haploid corn plant has 10 chromosomes, instead of 10pairs of chromosomes as in a normal diploid corn plant. A diploid planthas two sets of chromosomes and the chromosome number (2n) is equal tothat in the zygote. Each chromosome in a pair is derived from thematernal or paternal line.

A doubled haploid (“DH”) or a doubled haploid plant or cell refers tothe plant or its component developed by the doubling of a haploid set ofchromosomes. A doubled haploid plant is considered a homozygous plant. Aplant is considered to be doubled haploid even if the entire vegetativepart of the plant does not consist of the cells with the doubled set ofchromosomes, as long as the double haploid plant is fertile. The haploidplant may be derived from any plant and may be derived from aheterozygous plant. Such a heterozygous plant may or may not be a memberof a heterogeneous population of plants, such as from an open pollinatedpopulation of plants. The heterozygous plant may be obtained from anyheterozygous materials including local varieties, composites ofdifferent genetic backgrounds and other collections of plants.

A “haploid immature embryo” is defined as the embryo formed after onesperm nucleus from a pollen grain fuses with the polar nuclei in theembryo sac to create a triploid (3N) endosperm and before dry down.

A “doubled haploid embryo” is an embryo that has one or more cells thatcontain 2 sets of homozygous chromosomes. “Callus” refers to anundifferentiated proliferating mass of cells or tissue.

The phrase “contacting” includes reference to “direct contact” and“indirect contact”. For example, the medium comprising a doubling agentmay have direct contact with the haploid cell or the medium comprisingthe doubling agent may be separated from the haploid cell by a barrier,such as a filter paper, plant tissue, or other cells thus the doublingagent is transferred through the filter paper or cells or tissue to thehaploid cell. Contacting is achieved in any suitable manner, e.g.,hydroponic treatment of roots, spraying, injecting, infiltrating,soaking, and wetting.

The term “plant” includes reference to whole plants and plant components(e.g., leaves, stems, roots, pollen, etc.), seeds and plant cells andprogeny of same. “Plant cell,” as used herein, includes, withoutlimitation, seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. Plants that are capable of being used in the methods,disclosed herein, include both monocotyledonous and dicotyledonousplants.

Suitable plants include, for example, monocotyledonous plants, such as amaize, rice, wheat, barley, oats, onion, rye, millet, sorghum,sugarcane, lily or turfgrass plant.

The methods disclosed herein, generally produce a high frequency ofdoubled haploid plants that are fertile.

In an embodiment, methods to obtain a doubled haploid embryo, seed, orplant by contacting a haploid embryo with a doubling agent and obtaininga doubled haploid embryo, seed, or plant are disclosed.

In another embodiment, methods to obtain a doubled haploid cell culturefrom a suitable plant component, e.g., doubled haploid tissue cultureare disclosed.

A method to obtain a haploid plant component for inducing chromosomedoubling includes, for example, a) pollinating ovules, or stigmas, of aplant with pollen from an inducer line, wherein the inducer line has amarker gene that is expressed in embryos and/or endosperm tissue; and b)selecting a haploid embryo that has no marker gene expression.Pollination of the plant can be by any method, including naturalcross-pollination and manual pollination.

Somatic haploid cells, haploid embryos, haploid seeds, or haploidseedlings produced from haploid seeds can be treated with a lowmammalian toxicity chromosome doubling agent. Homozygous plants can beregenerated from haploid cells by contacting the haploid cells, such asembryo cells or callus produced from such cells, with chromosomedoubling agents, disclosed herein, to create homozygous doubled haploidcells. Treatment of a haploid seed or the resulting seedling or tissuegenerally may produce a chimeric plant or tissue, partially haploid andpartially doubled haploid. It may be beneficial to nick the seedlingbefore treatment with the doubling agent. When reproductive tissuecontains doubled haploid cells, then doubled haploid seed is produced.

Haploid embryos, haploid seeds, somatic haploid cells or tissue fromhaploid plants can be harvested and transformed by any known means priorto treatment with a doubling agent. Transgenic homozygous plants,regenerated from the transformed cells can be treated with a chromosomedoubling agent and growing the resulting seed/tissue to produce adoubled haploid plant having homozygous seeds.

In an aspect the inducer line may contain a scorable marker gene, forexample, colored markers in the endosperm, embryo, stem or leaves. Suchmarkers include GUS, luciferase, CRC, anthocyanin genes, such as A, C,R-nj, and others known in the art. When an inducer line with a scorablemarker is crossed with the selected/desired line, the resulting haploidseeds will have, for example, a colored endosperm with colorless embryo.It may be desirable to express the marker gene in the embryo. Inparticular, it may be desirable to express the marker gene in the earlystage of development, about 8-15 days after pollination using anappropriate promoter, such as an oleosin or a Lecl promoter. Markernegative embryos are then selected to obtain haploid embryos. Thismethod provides the advantage of obtaining haploid embryos withoutmarker genes. Similarly, haploid inducer lines, such as RWS, KEMS, RWS,ZMS or KMS, can be transformed with a lethal gene that is expressedspecifically in embryos of the mature seeds. After crossing the inducerline and the female parent the embryos of all the diploid seeds containthe inducible lethal gene, but the embryos of the haploid seeds do notcontain the lethal gene. The diploid F1 seeds cannot germinate due toexpression of the inducible lethal gene in their embryos. However, thehaploid seeds can germinate normally because they do not contain thelethal gene in their embryos. The germinating seedlings are haploid.

One or more genes of interest that are reflective of the commercialmarkets and interests of those involved in the development of crops canbe transformed or otherwise introduced into a line of interest prior togenerating a haploid seed or tissue by crossing with an inducer line.Such gene or genes of interest can also be transformed into a haploidcell or tissue prior to treatment with a chromosome doubling agent togenerate double haploid plant component.

General categories of genes of interest include, for example, includegenes encoding agronomic traits, insect resistance, disease resistance,herbicide resistance, sterility, grain characteristics, and thoseinvolved in oil, starch, carbohydrate, or nutrient metabolism as well asthose affecting, for example, kernel size, sucrose loading, and thelike.

Another method is obtaining a doubled haploid seed that includes: a)obtaining a haploid seed by pollinating an ovule with an inducer linewherein the ovule comprises a set of maternal chromosomes and whereinthe inducer line comprises a set of paternal chromosomes; b) contactingthe haploid seed with a medium comprising a low mammalian toxicitychromosome doubling agent; c) selecting a doubled haploid seed whereinthe doubled haploid seed comprises a triploid endosperm and a doubledhaploid embryo. The doubled haploid seed produced by such a methodwherein the triploid endosperm includes two sets of maternal chromosomesand one set of paternal chromosomes, and wherein the doubled haploidembryo has a first and second set of maternal chromosomes and whereinthe first set of maternal chromosomes is homozygous to the second set ofmaternal chromosomes can be produced.

In an embodiment, a method of obtaining a population of doubled haploidmaize plants include the following steps: a) obtaining a set of haploidkernels by pollinating an ear with an inducer line wherein the earincludes a set of maternal chromosomes and wherein the inducer line hasa set of paternal chromosomes; b) contacting the set of haploid kernelswith a medium having a low mammalian toxicity chromosome doubling agent;c) selecting a set of doubled haploid kernels wherein each kernel of theset of doubled haploid kernels includes a triploid endosperm and adoubled haploid embryo; d) growing the doubled haploid kernels into apopulation of doubled haploid maize plants. In other aspects, the earcan be removed before, during, or after pollination and placed into asolution. The doubling agent can come in contact with the ear afterpollination and before or after the ear is removed from the plant. Thechromosomal doubling agent may come into contact with the ear directlyor indirectly, for example, via filter paper or cotton or other suitablematerial.

A method of inbred selection may include the following steps: a)cross-pollinating two inbred maize plants; b) growing the F1 seed; c)pollinating the F1 plant with an inducer line to produce haploidembryos; d) contacting the haploid embryos with a low mammalian toxicitychromosome doubling agent to produce doubled haploid embryos; e)generating doubled haploid plants; f) analyzing the doubled haploidplants for agronomic performance. The development of haploids step mayalso be done at later generations, e.g., F2, F3, F4, etc. Producinghaploids from later generations allows for additional opportunities forrecombination, if needed.

If a haploid embryo is used as a source material for treatment with alow mammalian toxicity chromosome doubling agent, embryo rescue isperformed. Embryo rescue is performed by contacting an embryo with amedium containing nutrients and generating a plant. Phytohormones may ormay not be included in the embryo rescue medium. A method of obtaining atransgenic doubled haploid embryo may comprise isolating a haploidembryo, transforming the haploid embryo, placing the haploid embryo on amedium comprising a chromosome doubling agent and selecting a transgenicdoubled haploid embryo.

In any of these methods disclosed herein, the chromosomes can be doubledat the immature embryo stage, at the mature seed stage, or anytimebetween pollination of the plant and before the germination of thehaploid seed.

In any of these methods the haploid embryo that undergoes chromosomaldoubling may be isolated, may be in the seed or kernel, may be in thekernel on a slice of cob, may be on the ear or spike, or the haploidembryo may be in the kernel, which is on the ear and on the plant. Thedoubling agent may reach the haploid embryo while the ear is on theplant and the plant is intact. For example, the doubling agent may becontacted directly or indirectly with the haploid embryo. In some casesthe doubling agent can be transported by the plant. The plant may begrown hydroponically and the doubling agent can be taken up through theroots of the plant and transported to the haploid embryo. The seedlingsmay be initiated on soil or a growing medium and then transferred to ahydroponic solution where the doubling agent can be added. In anotheraspect of the method, the plant may be grown in soil or a growing mediumand then the doubling agent is added to the soil or growing medium sothat it can be transported to the haploid embryo.

The methods disclosed herein, avoid time consuming selfing and crossingmethods to obtain a homozygous trait of interest or a substantiallyhomozygous plant. The methods can be used to produce doubled haploidpopulations that do not contain the residual heterozygosity of inbredsobtained though the traditional method of self pollination. The methodscan be useful for functional genomics, such as knock-out analysis,functional analysis of recessive genes, gene replacement, homologousrecombination, gene targeting, transgene stacking, and evaluating lethalversus non-lethal analysis of genes. In addition, the chromosomedoubling agents, disclosed herein, are less toxic to mammalian systemscompared to the traditional agent, such as colchicine.

Haploid plant production systems have been developed and practiced forvarious plants to generate haploid tissues including embryo, plants andseeds. The haploid plants or plant components from any genotype aregenerated by crossing a selected/desirable line (as female) with aninducer line (male). Exemplary inducer lines for maize include, forexample, Stock 6 (Coe, 1959, Am. Nat. 93:381 382; Sharkar and Coe, 1966,Genetics 54:453 464) RWS, KEMS (Deimling, Roeber, and Geiger, 1997,Vortr. Pflanzenzuchtg 38:203 224), or KMS and ZMS (Chalyk, Bylich &Chebotar, 1994, MNL 68:47; Chalyk & Chebotar, 2000, Plant Breeding119:363 364), and indeterminate gametophyte (ig) mutation (Kermicle 1969Science 166:1422 1424). The disclosures of which are incorporated hereinby reference. Wide hybridization crosses can also be used to producehaploids. This method is sometimes referred to as the bulbosum method(Kasha and Kao, 1970, Nature 225:874-876). This method of haploidproduction occurs due to the elimination of the chromosomes from thepollinating parent.

EXAMPLES

The following examples are for illustrative purposes and are notintended to limit the scope of the disclosure. Those of skill in theart, in light of the present disclosure, would appreciate that manychanges can be made in the specific embodiments, which are disclosed andstill obtain a like or similar result without departing from the scopeof the disclosure.

Example 1 Generation of Double Haploid Plants in the Greenhouse Using1,2,3-Trimethoxy-4-((1S,6R)-6-nitro-cyclohex-3-enyl)-benzene(Compound 1) via Seedling Soaking

This example demonstrates the use of an aryl-substituted cycloalkene toobtain doubled haploid plants by a chromosome doubling approach. Anexemplary cycloalkene,1,2,3-Trimethoxy-4-(1S,6R)-6-nitro-cyclohex-3-enyl)-benzene, designatedas compound 1 was used to determine double haploid efficiency, doublingrate by a coleoptile tip cutting, seedling soaking method. Otherdoubling agents, such as colchicine, pronamide, oryzalin andchlorpropham were used for comparative purposes. Data provided in Tables1, 2 and 3 were generated using haploid seeds obtained through crosspollination of a maize line of interest with an inducer maize line.

The resulting maize seeds were sorted based on marker gene color.

Coleoptile tips in maize seedlings were cut and the seedlings weresoaked in various chromosome doubling agents as shown in Table 1.Haploid maize seeds were germinated and the coleoptile tips were excised(1-2 mm). The seedlings were then soaked in the specified concentrationsindicated in Table 1. Seedlings that survived were transplanted andgrown until tassels and silk formed. Plants that produced tassels andsilk were self-pollinated. Seeds produced from the self-pollination werevisually assessed for the marker to identify them as double haploid(dihaploid). For the cycloalkene designated as compound 1, the doublingrate was about 53% at 5 μM and 76.5% at 125 μM. The doubling rate of thetested cycloalkene is comparable to colchicines treatment at 0.06% w/v.Surprisingly, the mortality rate for compound 1 at 5 μM is only 15.1%(10/66*100) and even at a higher 25 μM concentration is only 30.3% ascompared to a mortality rate of 41.5% for colchicine.

Example 2 Generation of Double Haploid Plants in the Field Using1,2,3-Trimethoxy-4-((1S,6R)-6-nitro-cyclohex-3-enyl)-benzene(Compound 1) via Seedling Soaking

The procedure listed in example 1 was utilized to treat maize seedlingsthat were subsequently transplanted into the field. Seedlings thatsurvived the transplanting were grown to maturity. Plants that producedtassels and silk were self-pollinated. Seeds produced from theself-pollination were visually assessed for the marker to identify themas double haploid (dihaploid). Table 2 contains the data gathered forthe doubling rate and pollination success. For the 0.06% w/v colchicinetreatment, about 49 haploid plants were pollinated and about 38 plantshad doubled haploid ears, resulting in a success rate of about 77.5%(38/49*100). For the cycloalkene treatment, 25 μM and 100 μMconcentrations of compound Compound 1 was used. The 25 μM concentrationhad a success rate of 90% (36 out of 40 pollinated maize haploid plantshad doubled haploid ears). For the 100 μM treatment, 15 out of 24pollinated haploid maize plants had doubled haploid ears, resulting in asuccess rate of 62.5%.

Example 3 Generation of Double Haploid Plants Using1,2,3-Trimethoxy-4-(1S,6R)-6-nitro-cyclohex-3-enyl)-benzene (Compound 1)via Foliar Application

Haploid maize seeds were planted in Sun Gro METRO-MIX® 306 plantingmixture, which typically has a pH of 6.0 to 6.8 and an organic mattercontent of about 30 percent, in 72-well, plastic propagation flats(Dillen Products; Middlefield Ohio). When required to ensure goodgermination and healthy plants, a fungicide treatment and/or otherchemical or physical treatment was applied. The plants were grown in agreenhouse with an approximate 15 hour photoperiod which was maintainedat about 23-29° C. during the day and 22-28° C. during the night untilthey reached the V1-V3 growth stage. Nutrients and water were added on aregular basis and supplemental lighting was provided with overhead metalhalide 1000-Watt lamps as necessary.

A weighed amount of compound 1, determined by the rate to be tested, wasdissolved in 1.2 mL of dimethyl sulfoxide (DMSO) to obtain concentratedstock solutions. If the test compound did not dissolve readily, themixture was warmed and/or sonicated. The concentrated stock solutionsobtained were diluted with 10.8 mL of an aqueous mixture containing1.25% v/v (volume/volume) Agri-dex Crop Oil Concentrate. Compoundrequirements are based upon a 12 mL application volume at a rate of 187L/ha. Formulated compounds were applied to the plant material with anoverhead Mandel track sprayer equipped with a 8002E nozzle calibrated todeliver 187 L/ha over an application area of 0.503 square meter at aspray height of 18 inches (43 cm) above the average plant canopy height.Control plants were sprayed in the same manner with the solvent blank.

The treated plants and control plants were placed in a greenhouse, asdescribed above, and watered by sub-irrigation to prevent wash-off ofthe test compounds. Approximately 14 days after treatment, the treatedplants were transplanted to individual pots containing Sun GroMETRO-MIX® 360. Plants that produced tassels and silk were thenself-pollinated to obtain dihaploid seed.

Table 3 illustrates the results of doubling rate after spray treatmentwith the chemicals of interest. In comparison with the seedling soakingtreatment method, spray treatment of maize at the V1-V3 stage did notresult in significant plant stress, therefore, the corn plants grewnormally with no increase in mortality. The spray treated plant tasselsand silks were synchronized well and the plants shed more pollen. Thisresulted in a doubling rate from 31-46%.

In addition to the low mortality rates in plants, the cycloalkenedesignated as compound 1, exhibits low toxicity in mammalian systems.For example, cochicine shows oral LD50 (rat) of 5.89 mg/kg, whereascompound 1 shows LD₅₀ (rat) that is greater than or equal to 500 mg/kg.Eye irritation for colchicine is often observed for rabbit at about1%/3D and whereas for compound 1, in response to a dose of about 100mg/animal, 1 of 3 animals showed corneal and iridal effects at 24 hoursthat was resolved within 48 hrs. No dermal toxicity was observed forcompound 1 on rats at 200 mg/kg and no skin irritation was observed for250 mg/animal over a 7 day period on rabbits.

Therefore, this example demonstrates that the exemplary cycloalkene,1,2,3-Trimethoxy-4-((1S,6R)-6-nitro-cyclohex-3-enyl)-benzene, designatedas compound 1, functions as an effective chromosome doubling agent andresults in fewer deaths in plants when compared to conventional agents,such as colchicines.

TABLE 2 Doubled haploid success rates for various chemical treatments inthe field for maize. % haploid maize % DH plants pollinated pollinationTreatment (doubling rate) success 0.06% colchicine 55.68 77.55 Compound1, 25 μM 67.79 90 Compound 1, 100 μM 51.06 62.5 DH—doubled haploid

TABLE 3 Spray treatment of germinated haploid maize seedlings in soil atV1-V3 stage. # plants # plants rogued # plants Rate # of dead out (falsew/ (g ai/ seedings after haploid pollen % Treatment ha) sprayedtreatment seeds) and silk DR Compound 1 70 24 0 5 6 31.2 Compound 1 28033 0 7 12 46.2 Pronamide 35 19 0 1 5 27.8 Pronamide 140 22 0 4 3 16.7Chlorpropham 70 24 0 2 9 40.9 Chlorpropham 280 34 1 2 18 58.1DR-doubling rate

What is claimed is:
 1. A method for generating doubled haploid cornplant cells, the method comprising: contacting a leaf tissue of ahaploid corn plant, while the haploid corn plant is in a growth mediumat the growth stage of V1-V3, with a composition comprising a lowmammalian toxicity chromosome doubling agent to generate doubled haploidcorn plant cells; and generating the doubled haploid corn plant cells,wherein the low mammalian toxicity chromosome doubling agent comprises acompound of the formula:

wherein R¹, R², R³, R⁴, and R⁵ are in each instance independentlyselected from the group consisting of hydrogen, alkoxy, nitro, halo, andalkyl; A and B are each independently CH₂, CHR^(a) or CR^(a)R^(b),wherein R^(a) and R^(b) are in each instance independently selected fromthe group consisting of hydrogen, halogen, cyano, alkyl, alkenyl,alkoxy, haloalkyl, haloalkoxyl, alkylcarbonyl and alkoxycarbonyl, orR^(a) and R^(b) together with the attached carbon form a cycloalkylene;Z is CH═CH, CH₂—CH₂, CH₂═CH(CH₃), or CR^(d)═CR^(e), where R^(d) andR^(e) are each independently hydrogen, alkyl or R^(d) and R^(e) togetherwith the attached carbons form a cycloalkylene; and X and Y are eachindependently selected from the group consisting of hydrogen, NO₂, CN,halogen, alkylcarbonyl, alkoxycarbonyl, and CH₂—NO₂, provided that X andY are not both hydrogen.
 2. The method of claim 1, wherein the lowmammalian toxicity chromosome doubling agent is less toxic thancolchicine.
 3. The method of claim 1, wherein the low mammalian toxicitychromosome doubling agent has an oral LD₅₀ (rat) greater than 500 mg/kg.4. The method of claim 1, wherein the composition further comprisesdimethyl sulfoxide.
 5. The method of claim 1, wherein the compositionfurther comprises an agriculturally acceptable surfactant.
 6. The methodof claim 1, wherein the composition comprises an amount of the lowmammalian toxicity chromosome doubling agent effective to achieve achromosome doubling rate of from about 40% to about 75%.
 7. The methodof claim 1, wherein the amount of the chromosome doubling agent is fromabout 0.1 μM to about 200 μM.
 8. The method of claim 1, furthercomprising generating a whole doubled haploid corn plant from thedoubled haploid corn plant cells.
 9. The method of claim 8, furthercomprising generating doubled haploid corn seeds from the whole doubledhaploid corn plant.
 10. The method of claim 8, wherein the whole doubledhaploid corn plant produces a seed comprising a doubled haploid cornembryo and the doubled haploid corn embryo has a first and a second setof chromosomes that are homozygous.
 11. The method of claim 1, whereinthe leaf tissue of haploid corn plant is obtained from a haploid cornseed derived from a cross-pollination with an inducer line.
 12. (Themethod of claim 1, further comprising obtaining a population of haploidcorn plant cells by pollinating a corn ear with an inducer line.
 13. Themethod of claim 12, wherein the inducer line is selected from the groupconsisting of stock 6, stock 6 derivatives, stocks carryingindeterminate gametophyte (ig) mutation, RWS, KEMS, KMS, ZMS and MNL